Our armed forces are routinely exposed to hazardous weapons, pathogens, environmental toxins and, later, medical countermeasures with long-term health effects. The kidney is affected by many toxins metabolized in the body that it excretes, including products of rhabdomyolysis (skeletal muscle degradation), hemolysis, drugs, and exogenous toxins. Many of these compounds cause acute kidney injury (AKI) by producing reactive oxygen species (ROS), which activate apoptotic endonucleases. The resulting acute kidney failure (AKF) is a life-threatening condition that requires hemodialysis or kidney transplantation. This proposal is a continuation of the previous research funded by a VA Merit Review grant. The specific aims in the previous project have been accomplished and the new goals are logical extensions of these aims. The results from the previous study show that the importance of apoptotic endonuclease G (EndoG) in mediating myoglobinuric AKI is much more complex, not limited to DNA fragmentation only, and in some cases, completely opposite to what was initially accepted. The present proposal is based on our recent unexpected observations that myoglobinuric AKI is mediated by apoptotic deoxyribonuclease I (DNase I) regulated by EndoG. Prior to this study, no regulation of an endonuclease by another endonuclease has been described, and EndoG was considered one of the DNA-fragmenting apoptotic endonucleases. Contrary to this, we found that when EndoG becomes activated by later or stronger injury, it inactivates DNase I and thus protects against the injury. Therefore in the kidney, EndoG acts as cytoprotective enzyme instead of being cytotoxic. We hypothesize that myoglobinuric AKI can be prevented by inducing of EndoG-mediated inactivation of DNase I or otherwise inhibiting expression or activity of DNase I before and/or after injury. Our specific objectives are as follows. In Aim 1, we will evaluate native DNase I- and alternatively-spliced DNase I-based therapeutic approaches to ameliorate myoglobinuric tubular cell injury and AKI. In Aim 2, we plan to determine how EndoG-mediated inactivation of DNase I can be used to blunt myoglobunuric AKI. Aim 3 will be using our new high throughput technology to screen chemical library for new DNase I inhibitors applicable for the treatment of myoglobinuric AKI. Potential Impact on Veterans Health Care. Successful completion of these studies can potentially lead to the development of new therapeutic tools to prevent or ameliorate myoglobinuric AKI. Some of them will have strong translational value because they act even if administered after kidney injury, while others can become therapeutic options of the future. When applied to humans, the results of this study may allow saving human lives, improving the health of veterans, and decreasing the number of disabilities in the veteran population.